Development of a single culture E. coli expression system for the enzymatic synthesis of fluorinated tyrosine and its incorporation into proteins.

Current experiments that rely on biosynthetic metabolic protein labeling with 19F often require fluorinated amino acids, which in the case of 2- and 3-fluorotyrosine can be expensive. However, using these amino acids has provided valuable insight into protein dynamics, structure, and function. Here, we develop a new in-cell method for fluorinated tyrosine generation from readily available substituted phenols and subsequent metabolic labeling of proteins in a single bacterial expression culture. This approach uses a dual-gene plasmid encoding for a model protein BRD4(D1) and a tyrosine phenol lyase from Citrobacter freundii, which catalyzes the formation of tyrosine from phenol, pyruvate, and ammonium. Our system demonstrated both enzymatic fluorotyrosine production and expression of 19F-labeled proteins as analyzed by 19F NMR and LC-MS methods. Further optimization of our system should provide a cost-effective alternative to a variety of traditional protein-labeling strategies.

Complete genome sequences of Streptomyces spp. isolated from disease-suppressive soils.

BACKGROUND: Bacteria within the genus Streptomyces remain a major source of new natural product discovery and as soil inoculants in agriculture where they promote plant growth and protect from disease. Recently, Streptomyces spp. have been implicated as important members of naturally disease-suppressive soils. To shine more light on the ecology and evolution of disease-suppressive microbial communities, we have sequenced the genome of three Streptomyces strains isolated from disease-suppressive soils and compared them to previously sequenced isolates. Strains selected for sequencing had previously showed strong phenotypes in competition or signaling assays. RESULTS: Here we present the de novo sequencing of three strains of the genus Streptomyces isolated from disease-suppressive soils to produce high-quality complete genomes. Streptomyces sp. GS93-23, Streptomyces sp. 3211-3, and Streptomyces sp. S3-4 were found to have linear chromosomes of 8.24 Mb, 8.23 Mb, and greater than 7.5 Mb, respectively. In addition, two of the strains were found to have large, linear plasmids. Each strain harbors between 26 and 38 natural product biosynthetic gene clusters, on par with previously sequenced Streptomyces spp. We compared these newly sequenced genomes with those of previously sequenced organisms. We see substantial natural product biosynthetic diversity between closely related strains, with the gain/loss of episomal DNA elements being a primary driver of genome evolution. CONCLUSIONS: Long read sequencing data facilitates large contig assembly for high-GC Streptomyces genomes. While the sample number is too small for a definitive conclusion, we do not see evidence that disease suppressive soil isolates are particularly privileged in terms of numbers of biosynthetic gene clusters. The strong sequence similarity between GS93-23 and previously isolated Streptomyces lydicus suggests that species recruitment may contribute to the evolution of disease-suppressive microbial communities.

Simulation Modeling to Compare High-Throughput, Low-Iteration Optimization Strategies for Metabolic Engineering.

Increasing the final titer of a multi-gene metabolic pathway can be viewed as a multivariate optimization problem. While numerous multivariate optimization algorithms exist, few are specifically designed to accommodate the constraints posed by genetic engineering workflows. We present a strategy for optimizing expression levels across an arbitrary number of genes that requires few design-build-test iterations. We compare the performance of several optimization algorithms on a series of simulated expression landscapes. We show that optimal experimental design parameters depend on the degree of landscape ruggedness. This work provides a theoretical framework for designing and executing numerical optimization on multi-gene systems.

Engineering species-like barriers to sexual reproduction.

Controlling the exchange of genetic information between sexually reproducing populations has applications in agriculture, eradication of disease vectors, control of invasive species, and the safe study of emerging biotechnology applications. Here we introduce an approach to engineer a genetic barrier to sexual reproduction between otherwise compatible populations. Programmable transcription factors drive lethal gene expression in hybrid offspring following undesired mating events. As a proof of concept, we target the ACT1 promoter of the model organism Saccharomyces cerevisiae using a dCas9-based transcriptional activator. Lethal overexpression of actin results from mating this engineered strain with a strain containing the wild-type ACT1 promoter.Genetic isolation of a genetically modified organism represents a useful strategy for biocontainment. Here the authors use dCas9-VP64-driven gene expression to construct a 'species-like' barrier to reproduction between two otherwise compatible populations.

Genome Sequences for Streptomyces spp. Isolated from Disease-Suppressive Soils and Long-Term Ecological Research Sites.

We report here the high-quality genome sequences of three Streptomyces spp. isolated as part of a long-term study of microbial soil ecology. Streptomyces sp. strain GS93-23 was isolated from naturally disease-suppressive soil (DSS) in Grand Rapids, MN, and Streptomyces sp. strains S3-4 and 3211-3 were isolated from experimental plots in the Cedar Creek Ecosystem Science Reserve (CCESR).

Toward Bioremediation of Methylmercury Using Silica Encapsulated Escherichia coli Harboring the mer Operon.

Mercury is a highly toxic heavy metal and the ability of the neurotoxin methylmercury to biomagnify in the food chain is a serious concern for both public and environmental health globally. Because thousands of tons of mercury are released into the environment each year, remediation strategies are urgently needed and prompted this study. To facilitate remediation of both organic and inorganic forms of mercury, Escherichia coli was engineered to harbor a subset of genes (merRTPAB) from the mercury resistance operon. Protein products of the mer operon enable transport of mercury into the cell, cleavage of organic C-Hg bonds, and subsequent reduction of ionic mercury to the less toxic elemental form, Hg(0). E. coli containing merRTPAB was then encapsulated in silica beads resulting in a biological-based filtration material. Performing encapsulation in aerated mineral oil yielded silica beads that were smooth, spherical, and similar in diameter. Following encapsulation, E. coli containing merRTPAB retained the ability to degrade methylmercury and performed similarly to non-encapsulated cells. Due to the versatility of both the engineered mercury resistant strain and silica bead technology, this study provides a strong foundation for use of the resulting biological-based filtration material for methylmercury remediation.